PROJECT SUMMARY Currently over 5 million people are living with Alzheimer?s disease (AD) in the US. Unfortunately, no disease modifying therapies are yet approved, resulting in an enormous unmet medical need and burden on society. Genetic studies on patients with Late Onset AD (LOAD), which accounts for the vast majority of AD, point to defects in microglial function as the cause. Recently, we and others have identified mutations in the gene encoding TREM2 (triggering receptor expressed on myeloid cells 2) that correlate with a significantly increased risk of developing LOAD. In particular, the TREM2 R47H variant is associated with a risk similar to that of APOE4, previously the only well-established risk factor for LOAD. In the central nervous system, TREM2 is selectively expressed on microglia and is recognized to regulate the production of inflammatory cytokines, phagocytosis of apoptotic neurons and cell survival. TREM2 is a type 1 membrane protein of the Ig superfamily with a short cytoplasmic tail that interacts with?and signals through?DNAX-activating protein of 12 kDa (DAP12). In an exciting development, we and others have shown that TREM2 binds APOE and Clusterin (another apolipoprotein linked genetically to AD) and that mutations in TREM2 (e.g. R47H) reduce the binding of these ligands and also of oligomeric A?. Our current hypothesis is that impaired signaling through TREM2- DAP12 results in an altered immune response by microglia and this contributes to AD pathogenesis. In this revised application, we propose a high throughput screen of a large chemical library to identify compounds that bind to and modulate the function of TREM2 as a first step in translating these new biological insights into therapeutics. An innovative protein thermal shift (PTS) assay has been established that uses purified TREM2 to identify compounds that bind. This assay has been fully optimized in a 384 well format and as a demonstration of assay readiness, 45,000 compounds have been screened (Z?-parameter >0.7). Multiple hits from pilot screens were identified that dose dependently stabilized TREM2-but not control protein. A battery of downstream assays has been developed to establish a critical path-testing funnel. Specifically, orthogonal biophysical assays to confirm that hits physically bind TREM2 and secondary cellular assays to evaluate if hits affect the shedding of TREM2, alter the phosphorylation state of DAP12, and/or affect the activation state and properties of microglia. This proposal builds on data from the applicants, an established team from Sanford Burnham Prebys (Drs. Jackson, Sergienko and Xu) and Tanz CNRD, University of Toronto (Dr. St. George- Hyslop) who have been working together for the past 2 years, supported by philanthropic funds. The overall goal of this proposal is to generate a ?toolbox? of chemical probes (agonists, antagonists, allosteric modulators) that can be used to explore the biology of TREM2 and its role in LOAD. As the critical path assays are all in place, we anticipate we can rapidly obtain such probe molecules and start to explore their activity and ultimately their suitability for hit-to-lead and future in vivo evaluation in animal models and eventually patients.